Recognition of MHC-peptide complexes by the TCR activates Src and Syk family tyrosine kinases and induces phosphorylation of signaling proteins. One of the prominently phosphorylated proteins is LAT (linker for activation of T cells). LAT is a palmitoylated transmembrane adaptor protein that binds Grb2, Gads, PLC- y1, and other signaling molecules, thus recruiting these molecules to the plasma membrane to activate downstream signaling events, such as Ras-MAPK activation and Ca2+ flux. Previous studies with LAT- deficient Jurkat cells demonstrate that LAT is essential for TCP-mediated signal transduction and LAT palmitoylation is required for LAT phosphorylation and function. LAT-deficient mice have an early block in thymocyte development. Recent studies show that LAT also plays an important role in T cell homeostasis. Mice that express a LAT mutant that fails to bind PLC-y1 develop a severe autoimmune disease. Based on current information and our preliminary findings, we propose that, by coupling TCR engagement to Ras- MAPK activation and Ca2+ flux, LAT plays an important role in regulating T cell activation, survival, and homeostasis. There are three specific aims designed to test this hypothesis and to further understand how LAT functions in T cells. In specific aim 1, we will use mice, in which the Lat gene can be deleted by the Cre recombinase, to study the role of LAT during thymocyte development. In specific aim 2, we will induce deletion of the Lat gene in vivo and in vitro to study LAT function in mature T cells. We will focus on the role of LAT and the LAT-PLC? interaction in TCR-mediated signaling, T cell activation, homeostatic proliferation, and cell survival. In specific aim 3, we will use LAT conditional knockout mice to study whether normal LAT function is required for Treg cell survival in the periphery. We will also investigate the role of the Ca2+ pathway in Treg cell development and autoimmunity using mice that express a constitutively active form of calcineurin. T cells are the central components of our immune system. LAT is one of the molecules that play essential roles in T cell activation. Determination of LAT function and further understanding TCR signaling pathway could facilitate the design of a rational approach to augment or inhibit T cell proliferation in autoimmunity, allergy, and tissue and organ transplantation. [unreadable] [unreadable] [unreadable]